scholarly journals Far-red fluorescent proteins evolved from a blue chromoprotein from Actinia equina

2005 ◽  
Vol 392 (3) ◽  
pp. 649-654 ◽  
Author(s):  
Maria A. Shkrob ◽  
Yurii G. Yanushevich ◽  
Dmitriy M. Chudakov ◽  
Nadya G. Gurskaya ◽  
Yulii A. Labas ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Bathochromic Shift ◽  
Random Mutagenesis ◽  
Basal Disc ◽  
Red Fluorescent Proteins ◽  
Stable Mutant ◽  
Basal Disk ◽  
Green Fluorescent ◽  
Blue Circle

Proteins of the GFP (green fluorescent protein) family demonstrate a great spectral and phylogenetic diversity. However, there is still an intense demand for red-shifted GFP-like proteins in both basic and applied science. To obtain GFP-like chromoproteins with red-shifted absorption, we performed a broad search in blue-coloured Anthozoa species. We revealed specimens of Actinia equina (beadlet anemone) exhibiting a bright blue circle band at the edge of the basal disc. A novel blue chromoprotein, aeCP597, with an absorption maximum at 597 nm determining the coloration of the anemone basal disk was cloned. AeCP597 carries a chromophore chemically identical with that of the well-studied DsRed (red fluorescent protein from Discosoma sp.). Thus a strong 42-nm bathochromic shift of aeCP597 absorption compared with DsRed is determined by peculiarities of chromophore environment. Site-directed and random mutagenesis of aeCP597 resulted in far-red fluorescent mutants with emission maxima at up to 663 nm. The most bright and stable mutant AQ143 possessed excitation and emission maxima at 595 and 655 nm respectively. Thus aeCP597 and its fluorescent mutants set a new record of red-shifted absorption and emission maxima among GFP-like proteins.

Color morphs of the coral, Acropora tenuis, show different responses to environmental stress and different expression profiles of fluorescent-protein genes

2020 ◽  
Author(s):  
Noriyuki Satoh ◽  
Koji Kinjo ◽  
Kohei Shintaku ◽  
Daisuke Kezuka ◽  
Hiroo Ishimori ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Expression Profiles ◽  
Biological Significance ◽  
Gene Expression Profiles ◽  
Gfp Expression ◽  
Red Fluorescent Proteins ◽  
Color Morphs ◽  
Acropora Tenuis ◽  
Green Fluorescent

ABSTRACTCorals of the family Acroporidae are key structural components of reefs that support the most diverse marine ecosystems. Due to increasing anthropogenic stresses, coral reefs are in decline. Along the coast of Okinawa, Japan, three different color morphs of Acropora tenuis have been recognized for decades. These include brown (N morph), yellow-green (G) and purple (P) forms. The tips of axial coral polyps exhibit specific fluorescence spectra. This attribute is inherited asexually, and color morphs do not change seasonally. In Okinawa Prefecture, during the summer of 2017, the N and P morphs experienced bleaching, in which some N morphs died while P morphs recovered. In contrast, G morphs successfully withstood the stress. Symbiotic dinoflagellates are essential symbiotic partners of scleractinian corals. Photosynthetic activity of symbionts was reduced in July in N and P morphs; however, the three color-morphs host similar sets of Clade-C zoothanthellae, suggesting that beaching of N and P morphs cannot be attributed to differences in symbiont clades. The decoded Acropora tenuis genome includes five genes for green fluorescent proteins (GFP), two for cyan fluorescent proteins (CFP), three for red fluorescent proteins (RFP), and seven genes for chromoprotein (ChrP). A summer survey of gene expression profiles demonstrated that (a) expression of CFP and REP was quite low in all three morphs, (b) P morphs expressed higher levels of ChrP, (c) both N and G morphs expressed GFP highly, and (d) GFP expression was reduced in N morphs, compared to G morphs, which maintained higher levels of GFP expression throughout the summer. Although further studies are required to understand the biological significance of these color morphs of Acropora tenuis, our results suggest that thermal stress resistance is modified by genetic mechanisms that coincidentally lead to diversification of color morphs.

scholarly journals Incomplete proteasomal degradation of green fluorescent proteins in the context of tandem fluorescent protein timers

2015 ◽  
Author(s):  
Anton Khmelinskii ◽  
Matthias Meurer ◽  
Chi-Ting Ho ◽  
Birgit Besenbeck ◽  
Julia Fueller ◽  
...  
Keyword(s):  
Protein Turnover ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Proteasomal Degradation ◽  
Time Range ◽  
Green Fluorescent Proteins ◽  
Red Fluorescent Proteins ◽  
Dependent Change ◽  
The Stability ◽  
Green Fluorescent

Tandem fluorescent protein timers (tFTs) report on protein age through time-dependent change in color, which can be exploited to study protein turnover and trafficking. Each tFT, composed of two fluorescent proteins (FPs) that differ in maturation kinetics, is suited to follow protein dynamics within a specific time range determined by the maturation rates of both FPs. So far tFTs were constructed by combining different slower-maturing red fluorescent proteins (redFPs) with the same faster-maturing superfolder green fluorescent protein (sfGFP). Towards a comprehensive characterization of tFTs, we compare here tFTs composed of different faster-maturing greenFPs, while keeping the slower-maturing redFP constant (mCherry). Our results indicate that the greenFP maturation kinetics influences the time range of a tFT. Moreover, we observe that commonly used greenFPs can partially withstand proteasomal degradation due to the stability of the FP fold, which results in accumulation of tFT fragments in the cell. Depending on the order of FPs in the timer, incomplete proteasomal degradation either shifts the time range of the tFT towards slower time scales or precludes its use for measurements of protein turnover. We identify greenFPs that are efficiently degraded by the proteasome and provide simple guidelines for design of new tFTs.

scholarly journals Color morphs of the coral, Acropora tenuis, show different responses to environmental stress and different expression profiles of fluorescent-protein genes

2021 ◽  
Vol 11 (2) ◽  
Author(s):  
Noriyuki Satoh ◽  
Koji Kinjo ◽  
Kohei Shintaku ◽  
Daisuke Kezuka ◽  
Hiroo Ishimori ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Expression Profiles ◽  
Biological Significance ◽  
Gene Expression Profiles ◽  
Gfp Expression ◽  
Red Fluorescent Proteins ◽  
Color Morphs ◽  
Acropora Tenuis ◽  
Green Fluorescent

Abstract Corals of the family Acroporidae are key structural components of reefs that support the most diverse marine ecosystems. Due to increasing anthropogenic stresses, coral reefs are in decline. Along the coast of Okinawa, Japan, three different color morphs of Acropora tenuis have been recognized for decades. These include brown (N morph), yellow green (G), and purple (P) forms. The tips of axial polyps of each morph exhibit specific fluorescence spectra. This attribute is inherited asexually, and color morphs do not change seasonally. In Okinawa Prefecture, during the summer of 2017, N and P morphs experienced bleaching, in which many N morphs died. Dinoflagellates (Symbiodiniaceae) are essential partners of scleractinian corals, and photosynthetic activity of symbionts was reduced in N and P morphs. In contrast, G morphs successfully withstood the stress. Examination of the clade and type of Symbiodiniaceae indicated that the three color-morphs host similar sets of Clade-C symbionts, suggesting that beaching of N and P morphs is unlikely attributable to differences in the clade of Symbiodiniaceae the color morphs hosted. Fluorescent proteins play pivotal roles in physiological regulation of corals. Since the A. tenuis genome has been decoded, we identified five genes for green fluorescent proteins (GFPs), two for cyan fluorescent proteins (CFPs), three for red fluorescent proteins (RFPs), and seven genes for chromoprotein (ChrP). A summer survey of gene expression profiles under outdoor aquarium conditions demonstrated that (a) expression of CFP and REP was quite low during the summer in all three morphs, (b) P morphs expressed higher levels of ChrP than N and G morphs, (c) both N and G morphs expressed GFP more highly than P morphs, and (d) GFP expression in N morphs was reduced during summer whereas G morphs maintained high levels of GFP expression throughout the summer. Although further studies are required to understand the biological significance of these color morphs of A. tenuis, our results suggest that thermal stress resistance is modified by genetic mechanisms that coincidentally lead to diversification of color morphs of this coral.

scholarly journals A red fluorescent protein with improved monomericity enables ratiometric voltage imaging with ASAP3

2020 ◽  
Author(s):  
Benjamin B. Kim ◽  
Haodi Wu ◽  
Yukun A. Hao ◽  
Michael Pan ◽  
Mariya Chavarha ◽  
...  
Keyword(s):  
High Performance ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
High Gain ◽  
Motion Artifacts ◽  
Red Fluorescent Protein ◽  
Red Fluorescent Proteins ◽  
Transmembrane Voltage ◽  
Site Mutagenesis ◽  
Green Fluorescent

AbstractA ratiometric genetically encoded voltage indicator (GEVI) would be desirable for tracking transmembrane voltage changes in cells that are undergoing motion. To create a high-performance ratiometric GEVI, we explored the possibility of adding a voltage-independent red fluorophore to ASAP3, a high-gain green fluorescent GEVI. We performed combinatorial multi-site mutagenesis on the cyan-excitable red fluorescent protein mCyRFP1 to enhance brightness and monomericity, creating mCyRFP3. Among red fluorescent proteins tested, mCyRFP3 proved to be the least perturbing when fused to ASAP3. We demonstrate that the red fluorescence of ASAP3-mCyRFP3 (ASAP3-R3) provides an effective reference channel to remove motion artifacts from voltage-induced changes in green fluorescence. Finally we use ASAP3-R3 to visualize membrane voltage changes throughout the cell cycle of motile cells.

PROTONATION STATES AND CONFORMATIONAL FLEXIBILITY OF THE RED FLUORESCENT PROTEIN CHROMOPHORE

2009 ◽  
Vol 08 (06) ◽  
pp. 1117-1129 ◽  
Author(s):  
WEIZHONG YAN ◽  
DAIQIAN XIE ◽  
JUN ZENG
Keyword(s):  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Green Fluorescent Proteins ◽  
Minor Effect ◽  
Trans Isomerization ◽  
Red Fluorescent Proteins ◽  
Fluorescence Characteristics ◽  
Green Fluorescent ◽  
A Minor ◽  
Key Residues

Fluorescent proteins are important reporter molecules widely used in biotechnology and the biological sciences, generally. Their unusual spectrophotometric and fluorescence characteristics are controlled via protonation states of the chromophore, as for green fluorescent proteins (GFPs), and/or via cis–trans isomerization of the chromophore, as for red fluorescent proteins (RFPs). Here, we have performed both quantum mechanical calculations on substituted chromophores and structural comparisons of several RFPs (Rtms5, eqFP611, HcRed, and DsRed) and wild-type GFP. Our results indicate that the chromophore cis and trans isomers are comparably stable, and cis–trans isomerization has only a minor effect on electronic excitation. The chromophore is also found to exist in either the anionic or possibly zwitterionic protonation state. Structural comparisons of RFPs reveal that the conformation of the chromophore within a specific RFP is determined by only a few key residues, which could serve as mutation targets for engineering new fluorescent proteins for novel biotechnological applications.

scholarly journals Incomplete proteasomal degradation of green fluorescent proteins in the context of tandem fluorescent protein timers

2016 ◽  
Vol 27 (2) ◽  
pp. 360-370 ◽  
Author(s):  
Anton Khmelinskii ◽  
Matthias Meurer ◽  
Chi-Ting Ho ◽  
Birgit Besenbeck ◽  
Julia Füller ◽  
...  
Keyword(s):  
Protein Turnover ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Proteasomal Degradation ◽  
Time Range ◽  
Green Fluorescent Proteins ◽  
Red Fluorescent Proteins ◽  
Dependent Change ◽  
The Stability ◽  
Green Fluorescent

Tandem fluorescent protein timers (tFTs) report on protein age through time-dependent change in color, which can be exploited to study protein turnover and trafficking. Each tFT, composed of two fluorescent proteins (FPs) that differ in maturation kinetics, is suited to follow protein dynamics within a specific time range determined by the maturation rates of both FPs. So far, tFTs have been constructed by combining slower-maturing red fluorescent proteins (redFPs) with the faster-maturing superfolder green fluorescent protein (sfGFP). Toward a comprehensive characterization of tFTs, we compare here tFTs composed of different faster-maturing green fluorescent proteins (greenFPs) while keeping the slower-maturing redFP constant (mCherry). Our results indicate that the greenFP maturation kinetics influences the time range of a tFT. Moreover, we observe that commonly used greenFPs can partially withstand proteasomal degradation due to the stability of the FP fold, which results in accumulation of tFT fragments in the cell. Depending on the order of FPs in the timer, incomplete proteasomal degradation either shifts the time range of the tFT toward slower time scales or precludes its use for measurements of protein turnover. We identify greenFPs that are efficiently degraded by the proteasome and provide simple guidelines for the design of new tFTs.

Structural Evidence of Photoisomerization Pathways in Fluorescent Proteins

2019 ◽  
Author(s):  
Jeffrey Chang ◽  
Matthew Romei ◽  
Steven Boxer
Keyword(s):  
Rational Design ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Super Resolution ◽  
Unit Cell Size ◽  
Chlorine Substituent ◽  
Gfp Chromophore ◽  
The One ◽  
Green Fluorescent ◽  
Super Resolution Microscopy

<p>Double-bond photoisomerization in molecules such as the green fluorescent protein (GFP) chromophore can occur either via a volume-demanding one-bond-flip pathway or via a volume-conserving hula-twist pathway. Understanding the factors that determine the pathway of photoisomerization would inform the rational design of photoswitchable GFPs as improved tools for super-resolution microscopy. In this communication, we reveal the photoisomerization pathway of a photoswitchable GFP, rsEGFP2, by solving crystal structures of <i>cis</i> and <i>trans</i> rsEGFP2 containing a monochlorinated chromophore. The position of the chlorine substituent in the <i>trans</i> state breaks the symmetry of the phenolate ring of the chromophore and allows us to distinguish the two pathways. Surprisingly, we find that the pathway depends on the arrangement of protein monomers within the crystal lattice: in a looser packing, the one-bond-flip occurs, whereas in a tighter packing (7% smaller unit cell size), the hula-twist occurs.</p><p> </p><p> </p><p> </p><p> </p><p> </p><p> </p> <p> </p>

scholarly journals Split-wrmScarlet and split-sfGFP: Tools for faster, easier fluorescent l abeling of endogenous proteins in Caenorhabditis elegans

Genetics ◽  
2021 ◽  
Author(s):  
Jérôme Goudeau ◽  
Catherine S Sharp ◽  
Jonathan Paw ◽  
Laura Savy ◽  
Manuel D Leonetti ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Fluorescent Labeling ◽  
Large Fragment ◽  
C Elegans ◽  
High Affinity ◽  
Red Fluorescent Proteins ◽  
Invaluable Tool ◽  
Endogenous Proteins ◽  
Fluorescent Tags

Abstract We create and share a new red fluorophore, along with a set of strains, reagents and protocols, to make it faster and easier to label endogenous C. elegans proteins with fluorescent tags. CRISPR-mediated fluorescent labeling of C. elegans proteins is an invaluable tool, but it is much more difficult to insert fluorophore-size DNA segments than it is to make small gene edits. In principle, high-affinity asymmetrically split fluorescent proteins solve this problem in C. elegans: the small fragment can quickly and easily be fused to almost any protein of interest, and can be detected wherever the large fragment is expressed and complemented. However, there is currently only one available strain stably expressing the large fragment of a split fluorescent protein, restricting this solution to a single tissue (the germline) in the highly autofluorescent green channel. No available C. elegans lines express unbound large fragments of split red fluorescent proteins, and even state-of-the-art split red fluorescent proteins are dim compared to the canonical split-sfGFP protein. In this study, we engineer a bright, high-affinity new split red fluorophore, split-wrmScarlet. We generate transgenic C. elegans lines to allow easy single-color labeling in muscle or germline cells and dual-color labeling in somatic cells. We also describe a novel expression strategy for the germline, where traditional expression strategies struggle. We validate these strains by targeting split-wrmScarlet to several genes whose products label distinct organelles, and we provide a protocol for easy, cloning-free CRISPR/Cas9 editing. As the collection of split-FP strains for labeling in different tissues or organelles expands, we will post updates at doi.org/10.5281/zenodo.3993663

scholarly journals Multiphoton Bleaching of Red Fluorescent Proteins and the Ways to Reduce It

2022 ◽  
Vol 23 (2) ◽  
pp. 770
Author(s):  
Mikhail Drobizhev ◽  
Rosana S. Molina ◽  
Jacob Franklin
Keyword(s):  
Chemical Structure ◽  
Fluorescent Proteins ◽  
Multiphoton Ionization ◽  
Excitation Power ◽  
Photon Absorption ◽  
Green Fluorescent Proteins ◽  
Two Photon Absorption ◽  
Two Photon ◽  
Red Fluorescent Proteins ◽  
Green Fluorescent

Red fluorescent proteins and biosensors built upon them are potentially beneficial for two-photon laser microscopy (TPLM) because they can image deeper layers of tissue, compared to green fluorescent proteins. However, some publications report on their very fast photobleaching, especially upon excitation at 750–800 nm. Here we study the multiphoton bleaching properties of mCherry, mPlum, tdTomato, and jREX-GECO1, measuring power dependences of photobleaching rates K at different excitation wavelengths across the whole two-photon absorption spectrum. Although all these proteins contain the chromophore with the same chemical structure, the mechanisms of their multiphoton bleaching are different. The number of photons required to initiate a photochemical reaction varies, depending on wavelength and power, from 2 (all four proteins) to 3 (jREX-GECO1) to 4 (mCherry, mPlum, tdTomato), and even up to 8 (tdTomato). We found that at sufficiently low excitation power P, the rate K often follows a quadratic power dependence, that turns into higher order dependence (K~Pα with α > 2) when the power surpasses a particular threshold P*. An optimum intensity for TPLM is close to the P*, because it provides the highest signal-to-background ratio and any further reduction of laser intensity would not improve the fluorescence/bleaching rate ratio. Additionally, one should avoid using wavelengths shorter than a particular threshold to avoid fast bleaching due to multiphoton ionization.

scholarly journals Probing chemotaxis activity inEscherichia coliusing fluorescent protein fusions

2018 ◽  
Author(s):  
Clémence Roggo ◽  
Jan Roelof van der Meer
Keyword(s):  
Escherichia Coli ◽  
Protein Interactions ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Bacterial Chemotaxis ◽  
Ph Sensitive ◽  
Flagellar Motors ◽  
Receptor Interactions ◽  
Split Egfp ◽  
Green Fluorescent

ABSTRACTChemotaxis is based on ligand-receptor interactions that are transmitted via protein-protein interactions to the flagellar motors. Ligand-receptor interactions in chemotaxis can be deployed for the development of rapid biosensor assays, but there is no consensus as to what the best readout of such assays would have to be. Here we explore two potential fluorescent readouts of chemotactically activeEscherichia colicells. In the first, we probed interactions between the chemotaxis signaling proteins CheY and CheZ by fusing them individually with non-fluorescent parts of a ‘split’-Green Fluorescent Protein. Wild-type chemotactic cells but not mutants lacking the CheA kinase produced distinguishable fluorescence foci, two-thirds of which localize at the cell poles with the chemoreceptors and one-third at motor complexes. Cells expressing fusion proteins only were attracted to serine sources, demonstrating measurable functional interactions between CheY~P and CheZ. Fluorescent foci based on stable split-eGFP displayed small fluctuations in cells exposed to attractant or repellent, but those based on an unstable ASV-tagged eGFP showed a higher dynamic behaviour both in the foci intensity changes and the number of foci per cell. For the second readout, we expressed the pH-sensitive fluorophore pHluorin in the cyto- and periplasm of chemotactically activeE. coli. Calibrations of pHluorin fluorescence as a function of pH demonstrated that cells accumulating near a chemo-attractant temporally increase cytoplasmic pH while decreasing periplasmic pH. Both readouts thus show promise as proxies for chemotaxis activity, but will have to be further optimized in order to deliver practical biosensor assays.IMPORTANCEBacterial chemotaxis may be deployed for future biosensing purposes with the advantages of its chemoreceptor ligand-specificity and its minute-scale response time. On the downside, chemotaxis is ephemeral and more difficult to quantitatively read out than, e.g., reporter gene expression. It is thus important to investigate different alternative ways to interrogate chemotactic response of cells. Here we gauge the possibilities to measure dynamic response in theEscherichia colichemotaxis pathway resulting from phosphorylated CheY-CheZ interactions by using (unstable) split-fluorescent proteins. We further test whether pH differences between cyto- and periplasm as a result of chemotactic activity can be measured with help of pH-sensitive fluorescent proteins. Our results show that both approaches conceptually function, but will need further improvement in terms of detection and assay types to be practical for biosensing.

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